Abstract. A 32-year-old green-winged macaw (Ara chloroptera) was
diagnosed with chronic lymphocytic leukemia based on progressive
lymphocytosis and the presence of a monomorphic population of
well-differentiated lymphocytes in the bone marrow of a clinically
normal bird. Chemotherapy was initiated because of rapidly increasing
peripheral lymphocyte counts. In addition to oral prednisone (1 mg/kg
once daily), oral chlorambucil (1 mg/kg twice weekly) was initiated but
was discontinued after 6 weeks because of thrombocytopenia. The
leukocyte count was stabilized for 29 weeks with the concurrent use of
oral cyclophosphamide (5 mg/kg 4 d/wk) and daily prednisone, and the
bird exhibited a good quality of life. The bird died shortly after the
chemotherapy was inadvertently discontinued. The neoplastic cells from
this macaw stained positive for CD-3 antibody and negative for Bla.36,
suggesting the leukemia was of T-cell origin. This is the first report
of long-term treatment of a macaw with cyclophosphamide and documents
thrombocytopenia in a macaw secondary to chlorambucil, treatment.

Twenty-nine weeks after initial health examination the macaw was
anesthetized as before for physical examination, repeat CBC, and
whole-body radiographs. The bird weighed 1.15 kg, and results of the
physical examination were unremarkable. Ventrodorsal and lateral
radiographic examination revealed mild diffuse increased opacity in both
lungs. The only abnormality on the CBC was severe leukocytosis (65 000
cells/[micro]L) with lymphocytosis (59150 cells/[micro]L, 91% of
leukocytes). Evaluation of the blood smear was not recorded.

Three days later, bone marrow aspiration from the right tibiotarsus
was performed with the macaw under general anesthesia with isoflurane.
Cytologic examination of bone marrow aspirates stained with modified
Wright-Giemsa technique revealed a preponderance of small,
well-differentiated lymphocytes, few plasma cells, and very low numbers
of myeloid and erythroid precursors. The myeloid to erythroid ratio was
5 : 1, but only 25 hematopoietic cells could be found in the cytologic
preparations. The low hematopoietic cell count was attributed to
myelophthisis. These findings were consistent with a well-differentiated
lymphoid malignancy in the bone marrow and supported a diagnosis of
chronic lymphocytic leukemia (CLL).

Based on the elevated Aspergillus titer, aspergillosis was
suspected, and the bird was started on itraconazole (5 mg/kg PO q12h for
5 days, then q24h; Sporanox, Ortho-McNeil-Janssen Pharmaceuticals,
Titusville, NJ, USA). Because of the risk of immunosuppression, the bird
was also started on enrofloxacin (10 mg/kg PO q12h; Baytril 2.27%, Bayer
HealthCare LLC, Shawnee Mission, KS, USA) to cover the possibility of a
bacterial infection.

Thirty-four weeks after presentation and 7 weeks after the severe
leukocytosis was identified, the macaw was transported to the Louisiana
State University Veterinary Teaching Hospital for coelioscopy to
evaluate the bird for neoplastic or fungal masses. Anesthesia was
induced with isoflurane and maintained with isoflurane 2% administered
by endotracheal tube. Blood was collected from the right jugular vein
for a CBC and plasma biochemical analysis. Results revealed considerable
worsening of the leukocytosis (total WBC, 489000 cells/[micro]L),
primarily due to lymphocytosis (440 000 cells/[micro]L). Mild anemia
(packed cell volume [PCV] 36%, reference range, 45%-55%) was also
present. (1) A coelomic examination was performed with a 2.7-mm rigid
endoscope (Karl Storz Veterinary Endoscopy America Inc, Goleta, CA,
USA), on both cranial and caudal left thoracic air sacs and the left
abdominal air sac. Coelioscopy revealed pulmonary petechiation and
ecchymosis on the visceral side of the left lung. The air sac membranes
were slightly opaque, but no overt fungal plaques were observed. An
irregularly shaped, pale, highly vascular mass (size not recorded) was
visible near the caudo-ventral edge of the left lung and adjacent to the
proventriculus. Also, a large vascular structure (size not recorded) was
observed between the cranial and medial parts of the left kidney.
Because of the prominent vasculature associated with the masses, biopsy
was not attempted because of the risk of severe hemorrhage. Although the
kidney and liver were grossly considered normal, biopsy samples were
obtained from both organs. Tracheoscopy revealed no abnormalities, but a
deep tracheal swab was taken for fungal culture, which was negative. The
bird recovered uneventfully. Enrofloxacin was continued orally, and
piroxicam (0.75 mg/kg PO q12h for 3d; Feldene, Pfizer Inc, New York, NY,
USA) was initiated for its analgesic and antitumor properties. (4)

Based on the bird's peripheral lymphocytosis, the large
population of small, well-differentiated lymphocytes in the bone marrow,
and the absence of clinical signs, CLL was diagnosed. Chemotherapy was
started because of the rapid and dramatic increase in peripheral
lymphocyte counts, as well as the development of associated cytopenias
(anemia). Histologic examination of the hepatic and renal biopsy samples
revealed mononuclear cells whose exact lineage could not be determined
but which were suggestive of a lymphoid neoplasm. Because of the
positive Aspergillus titers, nebulization with clotrimazole (3 mL q24h;
clotrimazole topical solution 1% USP, Teva Pharmaceuticals USA,
Sellersville, PA, USA) was initiated 4 days after endoscopy. The bird
was exposed to the nebulized clotrimazole daily for 1 hour total.

Approximately 6 weeks after initiation of the
chlorambucil-prednisone treatment, the bird was restrained for routine
phlebotomy. Subjectively, hemostasis after phlebotomy was prolonged, and
thrombocyte count was low at 14000 cells/[micro]L (reference range,
20000-30 000 cells/[micro]L). (1,5) Based on the suspicion of
chlorambucil-induced thrombocytopenia, this drug was discontinued.
Prednisone was continued. The total WBC count at this time was 300 000
cells/[micro]L with lymphocytosis (294 000 cells/[micro]L, 98% of
leukocytes), and a mild anemia (PCV 40%; RBC count 2.25 x [10.sup.6]
cells/ [micro]L) was still present. Results of the biochemical analysis
were within reference ranges. Although still high, the gamma globulin
levels were lower (1.32 g/dL), and the A/G ratio of 0.75 was just below
the reference range.

One week after cessation of the chlorambucil, the bird was
anesthetized as before for phlebotomy and bone marrow aspiration to
evaluate the effect of treatment on the bone marrow. A noticeable
difference in hemostasis was observed, and the blood clotted immediately
after digital pressure was applied over the venipuncture site. Although
a thrombocyte count was not performed because of laboratory error,
adequate numbers of thrombocytes were observed by the laboratory. The
WBC count was 190 000 cells/[micro]L. Results of evaluation of the bone
marrow aspirate sample were similar to the previous sample, revealing
well-differentiated lymphoid cells suggestive of CLL.

Sixteen days after cessation of the chlorambucil, thrombocytes were
considered adequate (35 000 cells/[micro]L) and were similar in value to
a thrombocyte count in a healthy macaw at the same facility. The WBC
count was 250 000 cells/ [micro]L with 96% lymphocytes and 4%
heterophils. At approximately 46 weeks after presentation,
cyclophosphamide (5 mg/kg PO q24h for 4 days, repeat in 21 days;
Cytoxan, Bristol-Myers Squibb Company, Princeton, NJ, USA, compounded
into a 5 mg/mL suspension by Carr Drugs) was added to the prednisone
treatment; latex gloves were worn when handling the chemotherapeutic
agent. Because cyclophosphamide has been shown to cause hemorrhagic
cystitis in mammals, the bird was given lactated Ringer's solution
(25 mL SC q24h for 4 days) and furosemide (2 mg/ kg SC q24h for 4 days)
with the cyclophosphamide treatment to ensure proper diuresis. The daily
prednisone, itraconazole, and nebulized clotrimazole were continued.

Blood samples collected weekly over the next 3 weeks showed a mild
increase and then stabilization of the WBC count (275 000
cells/[micro]L, 325000 cells/[micro]L and 325000 cells/[micro]L; Fig 1).
Three weeks after the initial cyclophosphamide dose, it was reinitiated
q24h for 4 days and stopped for 3 days. This cycle of 4 days of
cyclophosphamide, 3 days without cyclophosphamide was continued weekly.
White blood cell counts were performed weekly, and biochemical analyses
were done monthly. During this time, the White blood cell count never
exceeded 375 000 cells/[micro]L (Fig 1), and thrombocyte counts were
normal. The bird tolerated the treatment well, maintained its weight,
and appeared to have a good quality of life.

[FIGURE 1 OMITTED]

Seventy-five weeks after initial presentation and 29 weeks after
the initiation of cyclophosphamide, Hurricane Katrina disrupted routine
husbandry and medical care at the zoological facility. The bird's
medications were discontinued for 5 weeks, at which point a physical
examination was performed. Despite being alert and responsive, the bird
exhibited mild paresis of the left leg. Palpation of limbs and full body
radiographs revealed no significant findings, and the paresis was
attributed to soft tissue trauma. Results of a CBC showed the WBC count
was extremely elevated (1 121 000 cells/[micro]L) with 98.5% lymphocytes
and anemia (PCV 28%). Five weeks after cessation of the bird's
medications, oral prednisone, cyclophosphamide, itraconazole, and
nebulized clotrimazole were restarted. Results of a CBC 1 week after the
medications were restarted revealed a less pronounced lymphocytic
leukocytosis (400 000 cells/[micro]L, 95% lymphocytes) and anemia (PCV
34%; RBC count 1.80 x [10.sup.6] cells/[micro]L). A biochemical analysis
was not performed.

Two weeks after initiation of the medications, the bird appeared
lethargic and weak with fluffed feathers and was admitted to the
hospital. Its weight was 0.9 kg, and the macaw was in thin body
condition, appeared dehydrated, and had poor feather quality. The liver
was palpably enlarged. The bird was perching but unable to balance if
moved. A blood sample obtained from the right basilic vein revealed
severe anemia (PCV 9%) and hyperglycemia (glucose, 495 mg/dL; reference
range, 200-350 mg/dL). (1) A CBC was not available due to laboratory
error. Treatment with cyclophosphamide, prednisone, itraconazole, and
nebulized clotrimazole was continued. The bird was started on
marbofloxacin (5 mg/kg PO q24h; Zeniquen, Pfizer Animal Health, Exton,
PA, USA) to treat a possible bacterial infection due to
immunosuppression from the CLL, and lactated Ringer's solution (25
mL SC once) and enteral nutrition (30 mL PO once; Emeraid Critical Care,
Lafeber Company, Cornell, IL, USA) were also administered. The bird died
before additional diagnostic tests could be pursued.

Necropsy revealed thickening of the caudal thoracic and abdominal
air sac membranes. The liver was enlarged and had small, disseminated
white foci. The great vessels at the base of the heart were enlarged.
There was no evidence of the masses previously observed during
coelioscopy. Histologically, the lymphoid malignancy was distributed
throughout the bone marrow, liver, pancreas, intestines, lung,
proventriculus, spleen, and air sacs. Solid foci of the neoplasm
comprised sheets of round ceils with small hyperchromatic nuclei
containing small nucleoli. Rare mitotic figures were present. The
neoplastic cells were occasionally admixed with low numbers of
well-differentiated plasma cells and Mott cells and scattered
histiocytes. The neoplastic cells stained uniformly and intensely
positive for CD3, a T-cell marker. The neoplastic cells stained
uniformly negative for Bla.36, a B-cell marker, although this marker did
stain the plasma cells and histiocytes in and around the neoplastic
population (Fig 2). In the small intestine, a focus of pseudomembrane
formation and ulceration associated with large numbers of large bacilli
was present in the mucosa adjacent to a focus of transmural ulceration
and neoplastic infiltration. The aorta had a partially occlusive and
recanalized thrombus. The heart had some mild interstitial fibrosis in
the ventricles and mild left ventricular myocardial necrosis.

Discussion

Lymphoid neoplasia is the most common form of hemolymphatic
neoplasia reported in avian species. (6-8) This type of neoplasia is
commonly diagnosed in poultry and has occasionally been reported in
mostly captive and companion birds of the orders Passeriformes,
Psittaciformes, and Columbiformes, (6,7,9) and in a small number of
raptors. (10-13) In psittacine birds, lymphoproliferative disorders are
not common in any species but have been described in budgerigars
(Melopsittacus undulatus), cockatoos (Cacatua species), an Amazon parrot
(Amazona ochrocephala oratrix), and macaws (Ara species). (5,7,14-17)
Lymphoproliferative tumors may manifest in the skin, (8,14,18) although
disseminated visceral disease (lymphoma) has also been reported.
(17,19,20) In most of these cases, treatment was not initiated or was
unsuccessful. To our knowledge, this is the first reported case of
long-term treatment of CLL in a macaw.

The clinical diagnosis of CLL in this 32-year-old male green-winged
macaw was made based on the abnormal hematologic results with
progressively increasing lymphocytic leukocytosis and anemia and the
presence of a monomorphic population of well-differentiated lymphocytes
in the bone marrow aspirate in a clinically normal bird. These
diagnostic criteria are extrapolated from the small animal literature.
Chronic lymphocytic leukemia is often diagnosed in asymptomatic domestic
mammals in which absolute peripheral lymphocytosis is the major
diagnostic abnormality and is associated with significant infiltration
of the bone marrow by well-differentiated lymphocytes. (21) The bird in
this report had a severe leukocytosis; the WBC count ranged from 20 000
to 1 121 000 cells/[micro]L with over 90% lymphocytes most of the time.
There is one report of a saker falcon (Falco cherrug) with lymphoid
leukemia in which the total WBC count was estimated at 1 700 000 to 1
800 000 cells/[micro]L, with 99% lymphocytes. (12) Other hematologic
abnormalities in domestic animals with CLL include anemia and
hyperglobulinemia, both of which were present in the green-winged macaw
with CLL. The bird in this case had a significant
hypergammaglobulinemia. Monoclonal gammopathies have been reported in
dogs with CLL in which serum is evaluated by protein electrophoresis.
(21) Monitoring results of protein electrophoresis can also aid in
evaluating response to treatment.

Lymphoid leukemia by definition is a tumor of peripheral blood and
bone marrow, as was the case in the macaw reported here. (6) This is in
contrast to lymphoma, which develops as a solid tumor in multiple
tissues and can involve the bone marrow. (6) Diagnosis of CLL in birds
can be challenging, particularly because this syndrome is poorly
characterized in the avian patient. The advanced stage of CLL at the
time of diagnosis may make it difficult to identify the origin of the
neoplasia in birds. By the time the disease is diagnosed, the neoplasia
may have disseminated to multiple organs including coelomic viscera and
may confound the determination of its origin. (6,10)

[FIGURE 2 OMITTED]

The positive CD3 immunohistochemical staining characteristics of
the neoplasm in this case suggest it was of T-cell origin. (19,22) The
neoplastic cells stained negative for Bla.36, a B-cell marker that has
also been shown to detect histiocytic cells in numerous species.
(8,18,23)

Avian lymphoproliferative disorders have been associated with
herpesvirus (Marek's disease) and retroviruses (avian leukosis
virus and reticuloendotheliosis virus), (10,18) Some lymphoid neoplasms
in psittacine birds are suspected to be caused by viruses because of the
resemblance to those syndromes caused by RNA viruses in domestic
chickens. However, there is currently no research proving this, (6,8)
and viral particles have not yet been demonstrated. (5,14,18) Viral
infection was not identified in this case based on negative serologic
and PCR testing for known psittacine viruses. (24,25)

Criteria for treatment of CLL include clinical signs of disease,
degree of increase in the peripheral lymphocyte count, evidence of organ
involvement, associated hematologic abnormalities such as anemia, and
rapid lymphocyte doubling time in less than 6 months. (21) Chemotherapy
was initiated in this case because of the dramatic lymphocytic
leukocytosis, the lymphocyte doubling time over a short time period,
anemia, and bone marrow involvement. In addition, lymphoid neoplasia
often leads to severe clinical disease in pet birds over time. (6) The
goal of CLL therapy in humans and small animals is to control the
disease and spare the bone marrow, as complete cures are rare. (21,26)
It may take more than a month for the hematologic and clinical
abnormalities to resolve. However, long survival times (more than 2
years in dogs) may be seen in animals with CLL even without treatment.
(21) Unfortunately, staging and grading schemes to direct treatment have
not been developed for avian species. (6) Dosages and regimens of
chemotherapeutic agents have been published with different results and
are usually derived from current small animal or human protocols,
although most are based on an alkylating agent paired with prednisone.
(5,8,11,12,17,27)

Chlorambucil was selected for its efficacy in treating CLLs in
humans, (26) cats, (21) dogs, (21) and birds, (5,8) with relatively few
reported toxicities. Chlorambucil is an alkylating agent with
immunosuppressant and antineoplastic properties. In one report, a pekin
duck (Anas platyrhynchos domesticus) with CLL was treated with
chlorambucil in combination with other chemotherapeutic agents, but the
bird died soon after treatment was initiated. (28) Thrombocytopenia was
observed in the duck during chemotherapy, but the cause was not
determined. A dosage of 2 mg/kg twice weekly has been used to
successfully treat cutaneous lymphosarcoma in an umbrella cockatoo
(Cacatua alba). (8) A dosage of 2 mg/kg chlorambucil once every 14 days
for 3 treatments was used to successfully treat a blue and gold macaw
(Ara ararauna) with cutaneous pseudolymphoma. (5) The authors reported
normal thrombocyte counts during the treatment period. Treatment with
chlorambucil at a dosage of 1 mg/kg twice weekly in this greenwinged
macaw was discontinued after 6 weeks because of prolonged hemostasis and
thrombocytopenia. Bone marrow suppression, thrombocytopenia, and
hepatotoxicity have been associated with chlorambucil administration in
other species. (28) In this case, no evidence of hepatotoxicity was
observed, and the biochemical results remained unremarkable. Birds with
lymphoid leukemia may have low thrombocyte counts, (6) although this
macaw's thrombocyte count returned to normal after stopping the
drug, suggesting chlorambucil may have contributed to thrombocytopenia
in this case. Because of the short duration of treatment, the efficacy
of the drug in this case is unknown.

Cyclophosphamide, a derivative of nitrogen mustard, was added to
the protocol treatment in lieu of chlorambucil based on small animal
protocols. (21) Cyclophosphamide was chosen because it is
platelet-sparing, readily available, and cost-effective, and because,
like chlorambucil, it is an alkylating agent. (29) In addition, it is
available as an oral formulation, which was important in a bird that was
not a candidate for repeated intravenous treatment. The bird tolerated
the drug well. A potential side effect of cyclophosphamide in small
animals is hemorrhagic cystitis, which is thought to be due to a direct
toxic effect of cyclophosphamide's metabolite, acrolein, on the
urinary tract mucosa. (30) In dogs receiving cyclophosphamide,
concurrent furosemide administration reduced the risk of sterile
hemorrhagic cystitis. (30) Because of this potential side effect, the
bird reported here was treated prophylactically with furosemide and
sub-cutaneous fluids. No adverse urinary effects were seen in this bird
with cyclophosphamide treatment, and the furosemide and fluid treatment
regimen frequency was reduced to every other cyclophosphamide treatment
because of contusions noted from multiple injections. Anatomic
differences between avian and mammalian urinary tracts or differences in
metabolism may make the urinary side effects of cyclophosphamide less
likely in avian patients.

Gastrointestinal toxicity was suspected in a report of intraosseous
cyclophosphamide administration in a Moluccan cockatoo (Cacatua
moluccensis). (17) The green-winged macaw in this case exhibited an
episode of regurgitation 1 day after initiation of oral
cyclophosphamide, but no additional gastrointestinal side effects were
observed. Cyclophosphamide treatment may have helped stabilize the WBC
count in this macaw. The bird's precipitous demise after cessation
of the chemotherapy for 5 weeks and the dramatic decrease in peripheral
lymphocyte count that was observed shortly before death when
cyclophosphamide therapy was restarted suggest that the chemotherapy
prevented progression of the leukemia.

Corticosteroids are commonly used in conjunction with an alkylating
agent to treat CLL because of their immunosuppressive and
lymphocytolytic properties. (21) The dosage of prednisone was similar to
that used in small animal chemotherapeutic protocols. (21) Adding
additional chemotherapeutic drugs, changing drugs, and increasing doses
would have been other options for intensifying treatment for the bird
had it survived.

The presumptive concurrent pulmonary aspergillosis based on
elevated titers was not confirmed on tracheal cultures or postmortem
examination. Aspergillosis can be challenging to diagnose in the avian
patient. False positive results can occur with serologic testing.
(31-33) Protein electrophoresis can aid in the diagnosis of
aspergillosis. The macaw had an increase in gamma globulin
concentrations and a reduced A/G ratio, both of which have been seen in
birds with aspergillosis. (32) Regardless, the patient was considered
susceptible to developing fulminant aspergillosis due to potential
immunosuppression from the CLL (6,10,34) and chemotherapy. In birds,
lymphoid tumors may cause immunosuppression by interfering with
cell-mediated and humoral immunity. (10)

This bird likely died of complications from advanced CLL, secondary
bacterial enteritis, and associated intestinal perforation. The
bacterial infection was likely precipitated by the intestinal neoplastic
infiltrate and immunosuppression. The anemia was most likely caused by
complete effacement of the bone marrow by the neoplastic cells, although
the chemotherapy and undocumented hemorrhage may have been contributing
factors. In future cases with avian patients on immunosuppressant drugs,
close monitoring for evidence of secondary bacterial or fungal
infections is warranted.

This case contributes to the relatively few reports of CLL and
long-term chemotherapy in birds. The green-winged macaw described here
was diagnosed with CLL based on profound peripheral lymphocytic
leukocytosis and supporting results of bone marrow cytology. Treatment
with chlorambucil resulted in thrombocytopenia, which resolved with
cessation of the drug. These observations suggest that thrombocyte
counts should be included in the monitoring of any bird treated with
chlorambucil, and thrombocytopenia might be avoided by a treatment
interval longer than 3.5 days. The bird was subsequently treated with
oral cyclophosphamide and prednisone for 29 weeks and maintained a good
quality of life. This case report supports the use of cyclophosphamide
and prednisone for CLL in birds, although more long-term studies are
needed to better define efficacy.

Acknowledgments. We thank the staff at the Audubon Zoo for their
dedicated care of this bird and the staff in the Exotic Animal Ward at
the Louisiana State University School of Veterinary Medicine for their
assistance with this case. We also thank Dr David Hunley for his
assistance with the chemotherapeutic regimen in this case. We also thank
the staff at Northwest ZooPath, including Jamie Kinion for data
retrieval and Christie Buie for image plating.